The polypeptide “Melanoma Inhibitory Activity” MIA, was discovered in 1989 as a factor that inhibits growth of melanoma tumor cells. The melanoma inhibitory activity (MIA) protein was identified within growth-inhibiting activities purified from tissue culture supernatant of the human melanoma cell line HTZ-19 (Bogdahn et al., Cancer Res. 1989; 49: 5358-5363). The antiproliferative action of MIA was also demonstrated in other tumor cells and Peripheral Blood Mononuclear Cells (Jachimczak et al., 2000, Proceeding of AACR, 41: 115).
Furthermore, in situ-hybridization experiments, as well as immuno histochemistry localize MIA in the developmental embryo within the growth zone of the skeletal system, and it is being expressed, secreted, and deposited around the chondrocytes.
In the developing mouse the expression is correlated with the forming of the skeletal system and is postnatal fading out with the exception of being expressed again during the maturation of the mammary system.
However, in the case of the tumors, MIA was found to be expressed and secreted into the serum by all of the malignant melanomas examined, but not in other skin tumor, including basal cell cancer and squamous cell cancer, nor in normal melanocytes and keratinocytes.
“Melanoma Inhibitory Activity”, MIA, is translated as a 131 amino acid precursor molecule and processed into a mature 107 amino acid protein after cleavage of a secretion signal. MIA provides clinically useful parameters in patients with Metastatic melanoma stages III and IV (Bosserhoff et al., Cancer Res. 1997; 57: 3149-3153; Bosserhoff et al., Hautarzt. 1998; 49: 762-769; Dreau et al., Oncol. Res. 1999; 11: 55-61; Deichmann et al., J. Clin. Oncol. 999; 17: 1891-1896). MIA was described to elicit antitumor activity by inhibiting proliferation of melanoma cell lines in vitro (Blesch et al., Cancer Res. 1994; 54: 5695-5701; Bogdahn et al., Cancer Res. 1989; 49: 5358-5363). However, further studies have revealed expression patterns inconsistent with a tumor suppressor. Expression of the wild-type MIA protein gene was not detected in normal skin and melanocytes, but was associated with progression of melanocytic tumors (Bosserhoff et al., Cancer Res. 1997; 57: 3149-3153; van Groningen et al., Cancer Res. 1995; 55: 6237-6243). More recently, it was suggested that the MIA protein specifically inhibits attachment of melanoma cells to fibronectin and laminin, thereby masking the binding site of integrins to these extracellular matrix (ECM) components and promoting invasion and metastasis in vivo (Bosserhoff et al., Cancer Res. 1997; 57: 3149-3153; Bosserhoff et al., J. Pathol. 1999; 187: 446-454; Guba et al., Br. J. Cancer 2000; 83: 1216-1222). Thus, the growth-inhibitory activity in vitro reflects the ability of the protein to interfere with the attachment of cell lines to the surface of tissue culture dishes in vitro (Blesch et al., Cancer Res. 1994; 54: 5695-5701).
Weilbach et al. (1990 Cancer Res. 50; 6981-86) further demonstrated that MIA inhibits cell proliferation by prolonging of the S-Phase and arrest of the cells in the G2 compartment.
Human rMIA inhibits IL-2- or PHA-induced Peripheral Blood Mononuclear Cells (PBMCs) proliferation in a dose-dependant manner. Additionally, auto- and allogenic LAK-cytotoxicity has been inhibited by MIA (Jachimczak et al., 2000, Proceeding of AACR, 41: 115).
Blesch et al. (1994 Cancer Res. 54; 5695-5701) confirmed that MIA acts as a potent tumor cell growth inhibitor for malignant melanoma cell and further extended this observation to other neuroectodermal tumors and concluded that MIA might be attractive as a future antitumor therapeutical substance.
The clinical correlation of MIA expression with melanoma progression was discovered by Bosserhoff et al. (1997, Cancer Res. 57; 3149-53; 1997, Anti-cancer Res. 19; 2691-3) showing enhanced MIA levels in 13-23% of stage I and II melanomas, but in 100% of stage III or stage IV disease.
Van Groningen et al. (1995 Cancer Res. 55; 6237-43) found MIA mRNA expression in non metastasising cell lines and an inverse correlation of MIA mRNA expression with pigmentation in melanoma metastasis lesions.
The 3D structure of the recombinant human MIA in solution was determined recently by multidimensional NMR spectroscopy and revealed that MIA is the first extracellular protein known to adopt an SH3 domain like-fold. These studies also provided evidence of specific interaction between a binding fold of MIA and a partial fibronectin peptide that has been implicated in integrin binding. It appears that MIA belongs to a growing family of proteins that promote invasion and metastasis by inhibiting specific interactions between integrins and ECM molecules within the local tumor milieu.
Furthermore, the 1.4 Angstrom resolution crystal structure of human MIA protein was determined by X-ray protein crystallography using multi-wavelength anomalous diffraction (MAD) (Lougheed et al., PNAS 2001 May 8; 98 (10):5515-5520). The structure confirms a conventional SH3-like fold of MIA.
The idea that proline-rich peptides are ligands of the SH3 domains has been supported by a number of experiments (for example see Ren et al., Science 259, 1157-1161, 1993; Gout et al., Cell 75, 25-26, 1993). Yu and coworkers (Cell 76, 933-945, 1994) have proposed that the specificity of SH3-ligand interactions may arise from the reciprocal recognition of non-proline peptide residues with non-conserved protein side chains. Musacchio et al. (Nat. Struct. Biol 1(8), 546-551, 1994) were unable to identify such specific interactions in complexes of SH3-domain proteins.
Lougheed et al. (PNAS 2001 May 8; 98 (10):5515-5520) tested the ability of MIA to bind proline-rich peptides by using phase display, but were unable to identify significant peptide binders out of a biased polyproline helix phage display library.
In contrast to these data the invention described below is based mainly on ligands with a high content of proline that do not possess a consenus sequence motif for binding to SH3 domains.